Therapeutic Effect Of Chai Huang Qing Yi Huo Xue Granule On Rats With Severe Acute Pancreatitis

Objective:To study the therapeutic effect of Chai Huang Qing Yi Huo Xue Granule(CHG)on rats with severe acute pancreatitis(SAP)and to provide experimental basis for the clinical application and further drug development of Chai Huang Qing Yi Huo Xue Granule.Methods:(1)Animal grouping:144healthy adult male SD rats were randomized to 6 groups: sham-operation group(Sham),SAP model group,low-,medium-,and high-dose Chai Huang Qing Yi Huo Xue Granule(ie,CHG1,CHG2,and CHG3)and positive control Qing Yi Li Dan Granule(QYG)with 24 rats in each group.(2)SAP model: The rat SAP model was prepared by retrograde injection of 3.5% sodium taurocholate(STC)in 1ml/kg through the duodenal papilla.The sham-operation group opened the abdomen.Only the abdominal organs were turned and then the abdominal cavity was closed.(3)Preparation of drug: The liquid extract of Chai Huang Qing Yi Huo Xue Granule was dissolved in distilled water at 90℃,and then formulated into a solution of 0.04g/ml,0.08g/ml,and 0.16g/ml,respectively.Qing Yi Li Dan Granule was dissolved in distilled water at 90℃ to be formulated as a 0.1 g/ml solution.The amount of delivered drug solution was calculated according to the weight of the rats to 10 ml/kg.(4)Dosing: After anesthesia,the low-,medium-,and high-dose groups of Chai Huang Qing Yi Huo Xue Granule were intragastrically administered with the drug solution containing 0.04 g/ml,0.08 g/ml,and 0.16 g/ml the liquid extract of Chai Huang Qing Yi Huo Xue Granule.Qing Yi Li Dan Granule group was treated with gavage of Qing Yi Li Dan Granule(0.1g/ml).The drug was delivered every 4hours.The sham-operation group and the SAP model group were given the same amount of normal saline.All rats were fasted after surgery and were free to water.(5)Material detection: There were 3 time points at 12 h,24h,and 48 h after operation.Eight rats were sacrificed in each group at each time point.After abdominal open anatomy,the ascites was collected by a combination of syringe suction and sterile cotton ball suction,and weighed.Blood was collected from the celiac artery.Whole blood white blood cell(WBC)counts were measured with the hematology analyzer.Serum amylase(AMY),lipase(LIP),aspartate aminotransferase(AST),alanine aminotransferase(ALT),creatine kinase-MB(CK-MB),blood urea nitrogen(BUN),blood glucose(GLU),and calcium ion(Ca2+)were measured using an automatic biochemical analyzer.Serum C-reactive protein(CRP)and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA).The pancreatic tissue was completely removed.The pancreatic head was weighed and dried in a constant-temperature drying oven at 60℃ for 48 hours until the weight of the pancreas changed no more.The pancreatic dry-to-wet ratio was calculated.The body of the pancreas was immersed in 4%neutral formaldehyde for 24 hours and then paraffin-coated and stained with haematoxylin-eosin staining(HE).The Kusske score method was used to analyze the histopathological score of the pancreas under light microscope.Results:(1)Compared with the sham-operation group,Gross anatomic observation of SAP model showed larger pancreatic tissue.The pancreas shows hyperemia,edema,hemorrhage,and necrosis with saponification spots,which were also found in mesenteries.Abdominal bloating and expansion was discovered with abundant yellow-green,reddish ascites and GI-produced gases,as well as the congested and edematous intestinal wall.HE staining of pancreatic tissue revealed structural disorder of the pancreas and even severe damage:acinar edema,enlarged interlobular septum,marked hemorrhage,massive necrosis and inflammatory cell infiltration.In the SAP model group,we observed a decreased dry-to-wet ration,increased histpathological score of the pancreas,increased activities of serum AMY and LIP,increased levels of the whole blood white blood cell count and serum CRP and TNF-α,decreased concentration of the calcium of ion,and increased concentration of the blood glucose.Impaired liver function(increased AST and ALT activity)and renal function(increased BUN concentration)were also observed.The differences were statistically significant(p<0.05),suggesting successful construction of rat SAP model.(2)Compared with the SAP model group,Chai Huang Qing Yi Huo Xue Granule of different dosage group and Qing Yi Li Dan Granule group showed slightly increased volume of the pancreas,hyperemia,edema and necrosis,sporadic saponification spot at overall anatomic observation.The degree of lesions was decreased compared with SAP model group.Especially in the high-dose group of Chai Huang Qing Yi Huo Xue Granule,the necrosis was small,scattered,and the ascites quantity was small.Pancreatic tissue stained with HE showed that the structure of the pancreas was normal,hyperemia and edema between the lobular,and a small amount of hemorrhage,focal necrosis,and a few infiltrated inflammatory cells.Chai Huang Qing Yi Huo Xue Granule of different dosage group and Qing Yi Li Dan Granule group can reduce pancreatic edema and histopathological score,decrease serum AMY,LIP activity,decrease whole blood white blood cell count and serum CRP,TNF-αlevels,increase calcium ion concentration,lower blood glucose concentration,protection of liver function(decreased AST and ALT activity),renal function(decreased BUN concentration).Compared with the SAP model group,thedifferences of the above indicators were statistically significant(p<0.05),suggesting that Chai Huang Qing Yi Huo Xue Granule has therapeutic effects on SAP rats.And its efficacy is dose-dependent.(3)Compared with the Qing Yi Li Dan Granule group,the differences of high dosage group of Chai Huang Qing Yi Huo Xue Granule in reducing the ascites quantity,pancreatic histopathological score,,white blood cell counts,serum CRP,serum TNF-α,and protecting liver function(decreased AST and ALT activity)were statistically significant(p<0.05),suggesting that the high-dose Chai Huang Qing Yi Huo Xue Granule had better therapeutic effects on SAP rats than Qing Yi Li Dan Granule.Conclusion:(1)Chai Huang Qing Yi Huo Xue Granule has therapeutic effects on rats with severe acute pancreatitis,and its efficacy is dose-dependent.(2)High-dose Chai Huang Qing Yi Huo Xue Granule had better therapeutic effect on rats with severe acute pancreatitis than Qing Yi Li Dan Granule.